Wang et al. BMC Medical Genetics(2020) 21:222
https://doi.org/10.1186/s12881-020-01157-0

CASE REPORTOpen Access

Prenatal diagnosis of Duchenne muscular
dystrophy revealed a novel mosaic
mutation in Dystrophin gene: a case report
Yan Wang1,2,3,4*†, Yuhan Chen1,2,3,5†, San Mei Wang1,2,3,5†, Xin Liu1,2,3,5, Ya Nan Gu1,2,3,5 and Zhichun Feng1,2,3,5*Abstracts
Background: Duchenne muscular dystrophies (DMDs) are X-linked recessive neuromuscular disorders withmalfunction or absence of the Dystrophin protein. Precise genetic diagnosis is critical for proper planning of patientcare and treatment. In this study, we described a Chinese family with mosaic DMD mutations and discussed thebest method for prenatal diagnosis and genetic counseling of X-linked familial disorders.
Methods: We investigated all variants of the whole dystrophin gene using multiple DNA samples isolated from theaffected family and identified two variants of the DMD gene in a sick boy and two female carriers by targeted next
generation sequencing (TNGS), Sanger sequencing, and haplotype analysis.
Results: We identified the hemizygous mutation c.6794delG (p.G2265Efs*6) of DMD in the sick boy, which wasinherited from his mother. Unexpectedly, a novel heterozygous mutation c.6796delA (p.I2266Ffs*5) of the samegene, which was considered to be a de novo variant, was detected from his younger sister instead of his mother
by Sanger sequencing. However, further NGS analysis of the mother and her amniotic fluid samples revealed thatthe mother carried a low-level mosaic c.6796delA mutation.
Conclusions: We reported two different mutations of the DMD gene in two siblings, including the novel mutation
c.6796delA (p.I2266Ffs*5) inherited from the asymptomatic mosaic-carrier mother. This finding has enriched theknowledge of the pathogenesis of DMD. If no mutation is detected in obligate carriers, the administration of
intricate STR/NGS/Sanger analysis will provide new ideas on the prenatal diagnosis of DMD.
Keywords: Duchenne muscular dystrophy, Prenatal diagnosis, Dystrophin gene, Next-generation sequencing,
Mosaicism

Background
Duchenne muscular dystrophy (DMD, OMIM #310200)
is a fatal X-linked recessive, inherited neuromusculardisorder characterized by muscle inflammation and progressive deterioration of muscle function [1–3]. The* Correspondence: 001wangyan@sina.com; zhichunfeng81@163.com†
Yan Wang, Yuhan Chen and San Mei Wang contributed equally to this work.
1
Department of Clinical Genetics, BaYi Children’s Hospital, Seventh Medical
Center of Chinese PLA General Hospital, Beijing 100700, China
2
National Engineering Laboratory for Birth defects prevention and control of
key technology, Beijing 100700, China
Full list of author information is available at the end of the article

estimated incidence ranges from 10.71 to 27.78 per 100,000 males [4, 5]. Patients with DMD primarily develop
muscle weakness between the ages of 2–5 years, during
which the symptoms slowly progress and render the patient immobile [6]. The mutation spectrum within thecausative gene Dystrophin is complex and varies in types,
sizes and locations, resulting in phenotype of different
scales [7, 8]. Considering the devastating effect of thisdisease and the lack of reliable treatments, early genetic
diagnosis and in depth prenatal screening are the mainmeans of reducing the disease incidence as well as of© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate ifchanges were made. The images or other third party material in this article are included in the article's Creative Commonslicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
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The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to thedata made available in this article, unless otherwise stated in a credit line to the data.

Wang et al. BMC Medical Genetics(2020) 21:222

further reducing alleviate severe permanent sequelae andeven death [9].
The classical genetic diagnosis of patients withclinically suspected DMD was conducted through a twostep procedure, including quantitative PCR based multiplex ligation-dependent probe amplification technology(MLPA) [10, 11] and Sanger sequencing [12, 13]. In recent years, benefiting from vast applications of NGS, an
increasing number of diseases with mosaic mutations invarious causative genes have been identified, such as
AKT1 in Proteus syndrome [14], IDH1 in Maffucci syndrome [15], DEPDC5 in cortical dysplasia-associatedepilepsy [16] and GNAS in McCune-Albright Syndrome[17]. In this study, we first described the hemizygous
mutation c.6794delG in the DMD gene of the proband
boy, which was inherited from the mother who carried
the heterozygous allele. Then we reported that his sister
had heterozygous variant c.6796delA, which was derived
from their mother, who was confirmed by NGS to carrya novel mosaic mutation c.6796delA (p.I2266Ffs*5) inthe same gene.

Case presentation
Patient and clinical evaluation

This study recruited a third-generation Chinese ancestry with 7 years of follow-up (Fig. 1). The

Fig. 1 Summary of the timeline of information reported in this case

Page 2 of 10

genealogical tree is shown in Fig. 2a. Our patient is
a 9-year-old Chinese boy (III: 7, Fig. 2a), born in
2003, the first child of healthy parents, who does nothave heredity history or consanguineous marriage.
He registered to our department of developmental
neurology in 2012 with clinical manifestations
similar to the phenotypic manifestations of DMD [6]including staggering gait, calf pseudo-hypertrophy,
inability to jump along with difficulties in runningor climbing, clumsy flat feet, Gowers’ sign on risingfrom floor, and obvious gross motor delay. Laboratory examinations showed significantly increased
level of serum Creatine Kinase (CK) (46,000 U/L).
The patient and his family were provided with careadvices including neuromuscular management,
rehabilitation management, gastrointestinal and nutritional management. In 2018, when his 34-year-old
mother was in the second trimester (II: 5), the family visited the Reproductive Medicine Center of our
hospital for prenatal consultation. The family history
survey informed us that the aunts and uncle of thepatient (II: 1, II: 3 and II: 7) shown in Fig. 2a were
all married and theiroffsprings are healthy.
Written consent from all guardians of the patient was
obtained. Informed consent from all individual participants included in the study was obtained.

Wang et al. BMC Medical Genetics

Fig. 2 (See legend on next page.)(2020) 21:222

Page 3 of 10

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Page 4 of 10(See figure on previous page.)
Fig. 2 Pedigree of the investigated family and its NGS results of DMD. a Genealogical tree of the DMD family. Proband, male
: Normal male;: Normal female;: Female DMD carrier;

DMD patient;: Female with unknown DMD. b Detection of dystrophin gene mutations by

MLPA (c) Detection of maternal components in the amniotic fluid using Microreader 21 Direct ID system. d The hemizygous mutation c.6794delG
was detected in the proband. e The heterozygous c.6796delA was detected in the amniotic fluid. f The frequency of c.6796delA detected in themother’s peripheral blood during her pregnancy was low, and the mosaic level was 3.87% (18/465). g The frequency of c.6796delA detected in
the mother’s peripheral blood after pregnancy was low, and the mosaic level was 5.31% (65/1224)

DNA contamination test

We initiated our study by specimen collection and DNA
extraction. We collected 3 ml peripheral blood andmixed it with EDTA-K2 for anticoagulation in designated family members. Hair follicle and oral swabs fromthe mother were also collected and DNA was extracted
from these samples for analysis. Under ultrasound guidance, amniotic membrane puncture was performed toobtain fetal exfoliated cells from the pregnant mother.
Total DNA of the indicated samples were extractedusing genomic DNA extraction kit according to theprocedures in previous publication [18]. The DNA concentrations of different samples were determined using
NANODROP™ 2000 instrument (Thermo Fisher Scientific, DE), and then the samples were stored at − 20 °C.
In order to eliminate the contamination of the amniotic fluid by the maternal blood at amniocentesis, DNA
samples from the mother (II: 5) and her amniotic fluid(III: 8) were amplified by Microreader 21 Direct ID
system (Microread, Suzhou China). It contains 13
combined DNA index system (CODIS) short tandem
repeat (STR) loci (CSF1PO, FGA, TH01, TPOX, vWA,
D3S1358, D5S818, D7S820, D8S1179, D13S317,
D16S539, D18S51, and D21S11), three expanded CODISSTR loci (D12S391, D19S433, and D2S1338), four nonCODIS STR loci (D6S1043, D2S441, Penta D, and PentaE), and one amelogenin locus in one reaction with afour-dye fluorescent (FAM, HEX, ROX and TAMRA)
analysis system. From the results in Fig. 2b, no maternal
DNA typing was found in the amniotic fluid, indicatingthat the amniotic fluid was not contaminated by maternal blood and could be used in subsequent tests. The
sex chromosomes of the amniotic fluid were X, X.

Detection of large mutations in dystrophin gene by MLPA

In order to determine whether the proband had large
rearrangements, MLPA was employed using the
SALSA MLPA probe mix P034-A3/P035-A3 DMD/
Becker (MRC Holland, Amsterdam, Netherlands), as
previously described [19]. As shown in Fig. 2c, no obvious deletion or duplication was detected. Similar
negative results were also obtained from the otherfamily members.

Mutation identification and validation by TNGS andsanger sequencing

The DNA samples of designated family members andthe amniotic fluid were quantified using NANODROP™
2000 instrument (Thermo Fisher Scientific, DE). Genomic DNA fragmentation and Library preparation werecarried out following the recommendations by Illumina
protocols. Sequence capture, enrichment, and elution
were performed according to the manufacturer’s instructions [20]. The captured fragments were sequenced inpair-end 150 bp mode using the Illumina HiSeq X ten
platform. Real Time Analysis (RTA) software v.1.8.70(Illumina) was used to analyze the image and obtain the
fluorescence intensity in sequences, and the FastQC
v0.10.1 program was used to control the data quality.
The clean reads were mapped to the UCSC hg19 humanreference genome using BWA. The Picard 1.114 tools
were used to remove the duplicated reads, and only theunique reads were used for variation detection. Subsequently, GATK Haplotype Caller was used to detect thevariants of SNPs and InDels, then GATK Variant Filtration was utilized to filter variants. After the above two
steps, data were transformed to VCF format. The variants were first annotated by ANNOVAR and prioritizedbased on the frequency of variants (MAF < 0.01) in 1000
genome, ESP6500, dbSNP, EXAC, HGMD, and further
predicted by SIFT, PolyPhen-2, Mutation Taster,
GERP++ and our in-house database.
In the current study, we used NGS to sequence a totalof 2.10 MB of the whole Dystrophin gene, including allexons, introns and promoter regions, which can help
provide the causative reason for DMD. Thorough NGS,a hemizygous variant was discovered from the probandof DMD (reference transcript: NM_004006.3) with thedeletion of a nucleotide G in exon 47, c.6794delG(p.G2265Efs*6) (Fig. 2d). In order to conduct genetic
counseling for the second child, DNA was extracted
from peripheral blood and amniotic fluid for sequencing.
The result of targeted sequencing indicated that theamniotic fluid carried another heterozygous mutation
c.6796delA (p.I2266Ffs*5) (Fig. 2e), and a low frequency(18/447, 3.87%) of this mutation was detected in the
peripheral blood of the mother in mid-pregnancy (Fig.
2f). When the child was delivered, the c.6796delA

Wang et al. BMC Medical Genetics(2020) 21:222

Page 5 of 10(p.I2266Ffs*5) mutation was re-examined and the lowlevel of mosaicism (65/1224, 5.31%) was confirmed (Fig.
2g). It is worth noting the c.6796delA variant has notbeen reported in any public databases or literature. In
addition, according to the American College of Medical
Genetics and Genomics (ACMG) interpretation of variants, this variant will result in a truncated protein and isclassified as “likely pathogenic”. All the high-throughput
sequencing results are summarized in Table 1.
Subsequently, the coding variations were validated bySanger sequencing. PCR amplification products were sequenced by Sangon Biotech Co. Ltd. (Shanghai, China)
to analyze the indicated DMD gene mutations in exon
47. The specific primers were designed by Primer
Premier 6 (http://www.premierbiosoft.com) and listed in
Supplementary Table 1. According to the family pedigree, the fact that the mother contained both c.6794G
heterozygous mutation and a low level of c.6796A mutation of Dystrophin on the same X chromosome
suggested that the mother could be a multi-site mosaicism carrier of DMD. This finding prompted us tofurther investigate. Importantly, these variants resulted
in the frameshift of amino acids 2265 and 2266, respectively. Both mutants are predicted to truncate DMD
translation by introducing a termination codon in exon
47 (Fig. 3a). The mutation of the proband c.6794delG(p.G2265Efs*6) resulted in frameshift and a prematurestop codon (Fig. 3b). Sequencing of the mother’s Dystrophin gene using DNA extracted from peripheral
blood revealed that the deletion of a heterozygous variant occurred at the same location (Fig. 3c). At the sametime, we found that the amniotic fluid sample (later born
as a daughter) carried another frameshift mutation
c.6796delA (p.I2266Ffs*5) in exon 47, which was a different heterozygous mutation from the mother and theproband (Fig. 3d). In order to further confirm the hypothesis of mosaicism, hair follicle and oral swab samples of the mother were characterized using Sanger
analysis and the results showed that the mutation wasc.6794delG. Therefore, considering the NGS data, thepossibility of mosaicism was preserved (Fig. 3e). In thiscase, no mutations of the Dystrophin gene were foundfrom the aunties of the proband (Fig. 3f, representative
image of elder aunty). In summary, the low mutation ratio of c.6796A detected by targeted sequencing analysis
can be attributed to the low level of mosaicism, which
was not easily confirmed by Sanger sequencing.
Determination of the origin of maternal chromosome

Through the sequence analysis of STR polymorphism
and SNP haplotype, we further determined the chromosomal origins of the above two mutations. For STR
analysis, primers were selected as described in the previous literature [21], and the detailed information is listed
in Fig. 4a. The five informative STRs (DXS9907, STR49,
DXS1067, DI623, 3′STR) were decisive for indirect identification of the high-risk haplotype in our case, theirlocus were located in intron 45, intron 49, intron 50, intron 62 and exon 79, respectively. The pedigree diagramof STR analysis is shown in Fig. 4b. By comparing thefragment size between the proband or amniotic fluid
and their mother, we confirmed the maternal origin of
the mutants. In addition, we conducted haplotype analysis consisting of 383 SNPs and discovered that thec.6794delG mutant monosomy in the proband and thec.6796delA mutant monosomy in the amniotic fluid
were inherited from the same X chromosome of themother (6 SNP sites in Fig. 4c and Supplementary
Table 2). In short, we successfully identified the pathogenic allele of the mutations.

Discussions and conclusions
In this study, we discovered a novel frameshift mutationof DMD, c.6796delA, using sequencing method. It is
worth noting that the proband carried a c.6794delG mutation in exon 47 of the Dystrophin gene, resulting in aframeshift and a premature stop codon (p.G2265Efs*6),
while the amniotic fluid sample (later born as daughter)
displayed different mutant site c.6796delA (p.I2266Ffs*5)in the same exon. Unexpectedly, we also identified alow-level mosaicism of the c.6796delA allele from themother who was already found to carry the c.6794delG
heterozygous mutation. Moreover, the fact that the firstreported c.6796delA mutation was initially undetected

Table 1 Deep sequencing of the family members in mosaic DMD cases
Sample Name

Mutant sites
ChrX-31,947,831 (c.6794delG)

ChrX-31,947,829 (c.6796delA)

Depth

Frequency (%)

ResultDepth

Frequency (%)

ResultProband522/568

91.90

Hemi

2/569

0.0

Non-carrier

Mother carrier(Pregnancy)

164/463

35.32

Het

18/4653.87

Low levelmosaicism

Amniotic Fluid

26/14350.0

Non-carrier

656/1446

45.36

Het

Mother carrier(Non-pregnancy)

517/1229

42.07

Het

65/1224

5.31

Low levelmosaicismWang et al. BMC Medical Genetics(2020) 21:222

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Fig. 3 Sanger analysis for the Dystrophin gene of the designated family members. a Overview of identified mutations. Note that c.6794delG
mutant generated a truncated dystrophin protein (p.Gly2265Glu Efs × 6), while c.6796delA mutant resulted in a truncated dystrophin protein(p.Ile2266phe Efs × 5). b-f Confirmation of the mutations by Sanger sequencing from the proband (b), the mother (c, e), the sister of the proband(d), and the aunty of the proband (f). Reference sequence for Dystrophin gene: NM_004006.3

by Sanger sequencing of maternal DNA broadened ourhorizon of the low-level mosaicism mechanism of thisapparent de novo mutation, implying that counselingand prenatal diagnosis for DMD should be conducted ina multi-dimensional and synergistic way.
Considering the complexity and randomness of themutation spectrum of DMD, numerous diagnostic
methods can be used to test DMD mutation, including

multiplex PCR [22], multiplex ligation dependent probe
amplification (MLPA) [23], array comparative genome
hybridization (array CGH) [24], PCR-based Sanger sequencing [25] and real-time PCR sequencing [26]. It isdifficult for clinical testing to cover the entire mutational
spectrum of DMD on a single platform. The targeted
NGS offers a comprehensive, accurate, rapid, and economical method to detect pathogenic mutations inWang et al. BMC Medical Genetics(2020) 21:222

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Fig. 4 Chromosome distribution of the two mutations. a Primers for the detection of DMD STR locus. b STR analysis result in the indicatedpedigree. c The sequencing result of the adjacent representative six SNPs near the mutation regions. Through the linkage genetic analysis using
informative STR and SNPs, it was deduced that the mutant allele was inherited from the same chromosome of the mother

DMD, which can help in providing both medical advices
to affected family members and prenatal diagnosis forthe mother of the proband [27, 28].
Importantly, mosaicism is a common but often
neglected condition that requires clinicians and geneticists to raise their awareness. Given the high proportion of mosaicism in apparent de novo cases of many
genetic disorders, failure to detect mosaicism in carriers could lead to serious consequences, because
their next child is at a high risk of carrying the same
mutation(s) [29, 30]. Previously, clinical evidences indicated that the higher transmitted rate of risk haplotypes in sporadic DMD cases may originate fromfemale carriers with germinal mosaicism [31, 32].

Therefore, in some cases with de novo mutations,prenatal analysis of the same genes should be considered because of a potential risk of mosaicism. In thisstudy, a parental mutation of c.6794delG was detectedin the first child delivered from a female carrier.
When carrier became pregnant again, the seemingly
high-risk mutation was not detected, but a truly novel
fetal mutation site, c.6796delA, emerged, which was
further confirmed when the child was born (Fig. 3).
The heterozygous mutation c.6796delA found in themother was identified to have low frequencies ofmosaicism during the middle-pregnancy and after thedelivery (3.87 and 5.31%, respectively). Therefore, theidentification of germline mosaicism reminds the

Wang et al. BMC Medical Genetics(2020) 21:222

affected families to conduct genetic counseling to understand the recurrence risk of the same disorders [33, 34].
So far, various therapeutic approaches have been extensively developed for DMD [35], most of which are
based on the mechanism of complex genetic mutations
to complementation or restoration of Dystrophin expression. It is worth noting that the full-length dystrophin protein contains four typical functional domains,
including the N terminal actin-binding Calponin
Homology (CH) domain (exon 1–8), the spectrin-richcentral Rod domain (exon 9–62), the Cystein Rich domain (exon 63–69), and C-terminal Zinc Finger domain(exon 70–79) (Fig. 5a) [36]. Importantly, there are two
genetic hotspots for deletion mutations in the DMD
gene, i.e., exons 45–55 and exons 3–7 [23]. These regions account for approximately 60 and 7% of the total
number of reported mutations in DMD, respectively(Fig. 5b) [37, 38]. The remaining mutations involve single nucleotide variants, small deletions or insertions,
single-base changes, and splice site changes [4, 39].
Compared to large deletion or duplications, the small insertions or substitutions are less common, and their distribution vary greatly. Interestingly, in the frequently
reported cases, the simple insertion or substitution

Page 8 of 10

mutations are more likely to resided in different Spectrins repeats within the Central Rod domain (Fig. 5c)[40–42], indicating that the possible significant
anchoring or binding functions are important for
maintaining the abundance and activity of dystrophy.
Indeed, Multiple-polymorphic sites were found within
the coding region of DMD and the reduced expression level of dystrophin is associated with the incidence of DMD [43]. Interestingly, the missense
mutation c.6794delG has been disclosed in a comprehensive study of DMD patients in South China [44].
In contrary, the novel missense mutation c.6796delA
is located in the rod domain and has not beenreported in any public variant databases, nor in any
chromosome from our in-house database. Insummary, all these results suggest that the first reported mutation c.6796delA might be a pathogenic
change. In our result, since both mutations arelocated in the 18th Spectrin region within the Rod
domain, the relationships between the mutations andthe function of Dystrophin are relatively poor (Fig.
5d). In the case of incomplete Rod structure, the detailed knowledge of how these frame shifts abolishedthe PPIs needs further study.

Fig. 5 Overview of the functional domains in the Dystrophin protein and the mutation hotspots. a In normal condition, the full-length protein
product is encoded by 79 exons and consists of four domains: N-terminal domain (ABD, green), internal rod-shaped domain (S, orange), cysteinerich domain (CR, pink) and Zinc Finger-rich carboxyl-terminal domain (ZF, red). b The dystrophin deletion hotspot regions, in which the N
terminal ABD and 45–55 exons in the central rod-like domain are gray. c SNV hotspot mutation regions are highlighted in red circle. d The
mutants discovered in our study are also highlighted

Wang et al. BMC Medical Genetics(2020) 21:222

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In clinical setting, genetic testing mostly focuses on
the proband and the parents. A negative result of mutation in the parents will inevitably lead to a conclusionthat the mutation starts from de novo. This tricky issue
can be resolved by offering prenatal diagnostic testingfor the pregnant woman who needs DMD counseling.
Since NGS is the only method which offers the mostcomprehensive analysis to reliably detect mosaic carriers,
prenatal analysis using NGS should be considered, especially in the cases of previously identified de novo
mutations.

Competing interests
The authors declare that they have no competing interests.

Supplementary Information

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The online version contains supplementary material available at https://doi.
org/10.1186/s12881-020-01157-0.
Additional file 1.
Additional file 2.

Abbreviations
DMD: Duchenne muscular dystrophy; NGS: Next generation sequencing;
SNP: Single nucleotide polymorphism; MLPA: Multiplex ligation-dependent
probe amplification; STR: Short tandem repeat; AKT1: AKT serine/threonine
kinase 1; IDH1: Isocitrate dehydrogenase (NADP(+)) 1 cytosolic; DEPDC5: DEP
domain containing 5; GNAS: GNAS complex locus
Acknowledgments
We thank all the tested individuals, their families and collaborating clinicians
for their participation. This study has neither been presented nor submittedor accepted anywhere.
Authors’ contributions
YW and ZCF conceived this study and takes responsibility for the quality ofthe data. YW and YHC acquired data and played important roles ininterpreting the results and writing the manuscript. SMW participated in thepatient sample collection and NGS data analysis. XL and YNG performed
data analysis. All authors have read and approved the final manuscript.
Funding
This research was supported by a grant (2017YFC1001700) of the National
Key Research and Development Program of China. These funds only
supported the cost of reagents for all genetics tests except for STR analysis.
They have no role in the design of the study and collection, analysis, andinterpretation of data and in drafting or polishing of the manuscript.
Availability of data and materials
The principal data generated and/or analyzed in this study are included in
the published article. The corresponding datasets are available from thecorresponding author on request.
Ethics approval and consent to participate
We ensure that written informed consent acknowledging their voluntary
involvement in the current study was obtained from all the patients
including the proband and his parents. All procedures performed in studies
involving human participants were in accordance with the ethical standardsof the Ethical Committee of the Seventh Medical Center of Chinese PLA
General Hospital and with the 1964 Helsinki declaration and its later
amendments or comparable ethical standards.
Consent for publication
The parents of the patients provided informed consent to publish this casereport, including case description, medical data, and images. Proof ofconsent to publish from study participants can be requested at any time anda copy of it is available to the journal.

Author details
1
Department of Clinical Genetics, BaYi Children’s Hospital, Seventh Medical
Center of Chinese PLA General Hospital, Beijing 100700, China. 2National
Engineering Laboratory for Birth defects prevention and control of key
technology, Beijing 100700, China. 3Beijing Key Laboratory of Pediatric Organ
Failure, Beijing 100700, China. 4Clinical Biobank Center, Chinese PLA General
Hospital, Beijing 100853, China. 5Department of Pediatrics, Chinese PLA
General Hospital, Beijing 100700, China.
Received: 18 August 2020 Accepted: 25 October 2020

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